For mammalian sperm to fertilize, they must gain the ability to move progressively, and then switch to a form of vigorous, non-progressive movement, or hyperactivation, as they near the site of fertilization. Our long-term goal is to understand the molecular basis for the different flagellar waveforms that typify these two types of motility. The driving premise of this proposal is that the mutant activity of the protein products of three tightly-linked t complex genes in sperm from t haplotype (t) homozygous males leads to expression of the abnormal flagellar waveform phenotype, "curlicue". In this phenotype, sperm briefly hyperactivate immediately upon release from the epididymides into a medium that supports fertilization, then suffer a sharp drop in curvilinear velocity accompanied by the chronic negative bending of the entire flagellum. As a result, t/t males are sterile. The three candidate proteins are: DNAHC8, a principal piece-restricted, axonemal dynein heavy chain with a unique N-terminus; TCTEX5, an inhibitory subunit of the sperm-restricted protein phosphatase, PP1gamma2, localizing primarily to the flagellar midpiece; and TSGA2, a testis/sperm-limited protein whose two major isoforms are differentially distributed in the cytoskeletal elements of both the principal piece and midpiece, and which may function as calcium-sensitive adaptor proteins. Thus, the t alleles of these three genes appear to specify defective components of pathways controlling flagellar waveform dynamics in mammalian sperm. We therefore propose three specific aims in which we will employ molecular/genetic, microscopic, and proteomic approaches to verify the efficacy and determine the role of each candidate gene product in the elaboration of "curlicue", based on comparative expression, localization, and protein-protein interaction studies of wild-type and t haplotype isoforms. These studies will improve our understanding of the complex molecular mechanisms that underlie abnormal movement of the mammalian sperm flagellum and other cilia in the body. In so doing, they will impact positively on our ability to identify novel therapeutic targets for diagnosing and treating human male infertility as well as other pathological conditions related to ciliary dysfunction. [unreadable] [unreadable] [unreadable]